All-Optical Miniaturized Co-culture Assay of Voltage-Gated Ca 2+ Channels

Methods Mol Biol. 2020;2173:247-260. doi: 10.1007/978-1-0716-0755-8_17.

Abstract

Light-activated proteins enable the reversible and spatiotemporal control of cellular events in optogenetics. Optogenetics is also rapidly expanding into the field of drug discovery where it provides cost-effective and noninvasive approaches for cell manipulation in high-throughput screens. Here, we present a prototypical cell-based assay that applies Channelrhodopsin2 (ChR2) to recapitulate physiological membrane potential changes and test for voltage-gated ion channel (VGIC) blockade. ChR2 and the voltage-gated Ca2+ channel 1.2 (CaV1.2) are expressed in individual HEK293 cell lines that are then co-cultured for formation of gap junctions and an electrical syncytium. This co-culture allows identification of blockers using parallel fluorescence plate readers in the 384-well plate format in an all-optical mode of operation. The assay is transferable to other VGICs by modularly combining new and existing cell lines and potentially also to other drug targets.

Keywords: 384-well plate; All-optical; CaV1.2; Channelrhodopsin; FLIPR; High-throughput screening; Optogenetics; Syncytium; Voltage-gated ion channel.

MeSH terms

  • Calcium Channels, L-Type / genetics
  • Calcium Channels, L-Type / metabolism*
  • Cell Line
  • Cells, Cultured
  • Channelrhodopsins / genetics
  • Channelrhodopsins / metabolism
  • Coculture Techniques
  • Giant Cells / metabolism
  • HEK293 Cells
  • Humans
  • Optogenetics / methods*
  • Patch-Clamp Techniques

Substances

  • Calcium Channels, L-Type
  • Channelrhodopsins
  • L-type calcium channel alpha(1C)